Protein nectin-3

ABSTRACT

This application provides a novel protein, nectin-3 having an amino acid sequence of any of SEQ ID NO: 2, 4 or 6, which belongs to a nectin protein family participating in cell-cell adhesion. It also provides a polynucleotide that codes for the nectin-3 and has a base sequence of any of SEQ ID NO: 1, 3 or 5; a recombinant vector having the polynucleotide; and an antibody against the protein nectin-3. The protein nectin-3 provides important information for clarifying all aspects of the molecular mechanism in cell-cell binding systems, and, in addition, it leads to the possibility of clarifying the mechanism of, for example, humectation and metastasis of carcinoma, and is expected to be applicable to diagnosis of carcinoma for its malignancy and to a method for treating cases with carcinoma and also to development of medicines for carcinoma.

TECHNICAL FIELD

[0001] The invention of this application relates to a novel proteinnectin-3 that participates in cadherin-based cell-cell adherensjunctions, and to gene-engineering materials for obtaining and utilizingthe protein.

BACKGROUND ART

[0002] Cell-cell adhesion systems to be formed by transmembraneproteins, such as adhesion molecules, receptors and channels play animportant role in various cell-level phenomena such as cell-celladhesion, cell movement and cell morphology determination in animalindividuals. Above all, cell-cell adherens junctions (AJs) bear a roleindispensable for histocompatibility. Evidence is increasing to say thatAJs further participate in controlling cell propagation and morphologictissue formation, in addition to their mechanistic role as above, It hasbecome clarified that many F-actin-binding proteins play a role as alinker to link actin cytoskeletons to adhesion molecules. However, themolecule-level understanding of AJs is insufficient, and it is not clearas to which molecules may bind actin cytoskeletons to cell membranes.

[0003] To clarify this, the inventors of this application have isolatedsome novel F-actin-binding proteins from rats' brains, analyzed thestructure of the protein especially specific to neurocytes andabundantly existing in synapses, and named it “neurabin”, for which theapplicant already filed a patent application (Japanese PatentApplication No. 276784/1998). Further, the inventors of this applicationhave individually isolated 1-afadin, a novel F-actin-binding protein,and ponsin, a protein that binds with 1-afadin, for both of which theapplicant filed patent applications (for 1-afadin, Japanese PatentApplication No. 89572/1999; for ponsin, Japanese Patent Application No.174687/1999). Still further, the inventors of this application haveidentified novel proteins, nectin-1α, 1β and nectin-2α, 2δ that functionfor cadherin-based AJ formation along with afadin (J. Cell Biol.,145:539-549, 1999). They have found that these nectin-1, -2 areCa²⁺-independent immunoglobulin-like adhesion molecules and act on AJformation in one system along with afadin and ponsin (Genes Cells4:573-581, 1999; J. Biol. Chem., 275:613-618, 2000).

[0004] Clarifying the molecular mechanism of cell-cell adhesion willmake it possible to clarify, for example, the mechanism of humectationand metastasis of carcinoma, and is expected to be applicable todiagnosis of carcinoma and also to development of medicines forcarcinoma. For clarifying it, it is necessary to clarify all the detailsof the molecules that participate in cell-cell adhesion.

[0005] The invention of this application has been made in considerationof the current situation as above, and its one object is to provide anovel protein that participates in cell-cell adherens junctions.

[0006] Another object of the invention is to provide a gene-engineeringmaterial for producing the protein.

DISCLOSURE OF THE INVENTION

[0007] To solve the problems noted above, this application provides aprotein nectin-3 having the amino acid sequence of any of SEQ ID NO:2, 4or 6.

[0008] The protein nectin-3 is derived from mice, including threesplicing variants expressed by mouse genomic gene (these proteins arehereinafter referred to as nectin-3α, nectin-3β, nectin 3γ.

[0009] This application also provides a polynucleotide that encodes theprotein nectin-3. The polynucleotide includes genomic DNA, mRNAs andcDNAs.

[0010] This application further provides cDNAs each encoding the threetypes of nectin-3, or that is, polynucleotides each having the basesequence of any of SEQ ID NO: 1, 3 or 5.

[0011] This application still further provides a recombinant vectorhaving any of these polynucleotides.

[0012] This application still further provides an antibody against anyof the three types of protein nectin-3.

[0013] Like nectin-1 and nectin-2, the protein nectin-3 of thisinvention is common to all mammals, existing in any and every mammal.Therefore, the protein nectin-3 and the polynucleotide encoding it arenot limited to only mouse-derived ones.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is to compare the amino acid sequences of nectin-3α, -3βand -3γ. The black background indicates the identical sequence among thethree variants; the gray background indicates the identical sequencebetween nectin-3β and -3γ; the underline indicates the signal peptide;the double underlines indicate the transmembrane regions; and theasterisks indicate the glycosylated sites in asparagine.

[0015]FIG. 2 shows the experimental data of trans homo-interaction ofnectin-3α. (A) indicates the expression level of nectin-3α, for which acell lysate was subjected to development in SDS-PAGE followed by Westernblot analysis with a polyclonal anti-nectin-3α antibody. (B) indicatescell aggregation activities of parental L cells (o) andnectin-3α-expressing L cells (). (C) indicates cell aggregationactivities of parental L cells (C1) and nectin-3α-expressing L cells(C2). The bar is 100 μm.

[0016]FIG. 3 shows the experimental data of cis homo-dimerization ofnectin 3α. Nectin-3α-expressing cells were incubated in the presence orabsence of BS3; each cell lysate was subjected to development inSDS-PAGE followed by Western blot analysis with a polyclonalanti-nectin-3α antibody. The arrow indicates the monomer; and thearrowhead indicates the dimer.

[0017]FIG. 4 shows the experimental data of mixed-cell aggregationactivities of labeled nectin-1α-expressing L cells and unlabelednectin-2α-expressing L cells (A1 to A3), labeled nectin-3α-expressing Lcells and unlabeled nectin-1α-expressing L cells (B1 to B3), and labelednectin-3α-expressing L cells and unlabeled nectin-2α-expressing L cells(C1 to C3). A1, B1 and C1 are the images in interference contrastmicroscopy; A2, B2 and C2 are the images in flourescence microscopy; A3,B3 and C3 are the data in statistical analysis. The bars are 40 μm.

[0018]FIG. 5 shows the images in immunofluorescence microscopy ofco-cultured two L cell lines of nectin-1α-expressing L cells and-2α-expressing L cells (A1 to A3), nectin-3α-expressing L cells and-1α-expressing L cells (B1 to B3), and nectin-3α-expressing L cells and-2α-expressing L cells (C1 to C3). In A1 to B2, seen is nectin-1α; in A2and C2, nectin-2α; in B1, nectin-3α (monoclonal antibody); in C1,nectin-3α (polyclonal antibody); and in A3, B3 and C3, merged. The barsare 10 μm.

[0019]FIG. 6 shows the experimental data of trans hetero-interactionaffinity, for which a cell composition (labeled and unlabeled cells) oftwo-cell aggregates was quantitatively analyzed. A indicates the data oflabeled nectin-1α-expressing L cells and unlabeled nectin-2α-expressingL cells; B indicates the data of labeled nectin-3α-expressing L cellsand unlabeled nectin-1α-expressing L cells; and C indicates the data oflabeled nectin-3α-expressing L cells and unlabeled nectin-2α-expressingL cells.

[0020]FIG. 7 shows the experimental data of cis hetero-dimerizationnectin 3α with nectin-1α or -2α. A is for comparison of cisdimerization. A1 indicates the data of nectin-1α-expressing L cells(lane 1) and -1α/3α-expressing L cells (lane 2); A2 indicates the dataof nectin-2α-expressing L cells (lane 1) and -2α/3α-expressing L cells(lane 2). B shows the data of immunoprecipitation. B1 indicates the dataof nectin-1α/3α-expressing L cells; B2 indicates the data ofnectin-2α/3α-expressing L cells. The cell extract is in lane 1; thesupernatant is in lane 2; the precipitate is in lane 3. The arrowsindicate monomers; and the arrowheads indicate the dimers.

[0021]FIG. 8 shows the experimental data of nectin-3 distribution intissues. A shows the data of Northern blot analysis of nectin-1 (A1),nectin-2 (A2) and nectin-3 (A3). B shows the data of Northern blotanalysis of nectin-3α (B1), nectin 3β (B2) and nectin-3γ (B3). Lane 1 isin heart; lane 2 is in brain; lane 3 is in spleen; lane 4 is in lung;lane 5 is in liver; lane 6 is in skeletal muscle; lane 7 is in kidney;and lane 8 is in testis.

[0022]FIG. 9 shows the experimental data of intracellular localizationof nectin-3α in mouse small intestine absorptive epithelia. In A, seenis nectin-3α; in B, nectin-2; and in C, merged. The asterisks indicatethe inner space of small intestines. The bar is 10 μm.

[0023]FIG. 10 shows the experimental data of direct binding of afadin tonectin-3α. The arrow indicates a fusion protein, GST-nectin-3α-CP; andthe arrowhead indicates a fusion protein, MBP-afadin-PDZ.

BEST MODE FOR CARRYING OUT THE INVENTION

[0024] A mouse protein, nectin-3α is encoded by 1650 bp cDNA having thebase sequence of SEQ ID NO: 1, and this has the sequence of 549 aminoacids as in SEQ ID NO: 2. The complete cDNA of the nectin-3α has thebase sequence of 2178 bp as in SEQ ID NO: 7.

[0025] Nectin-3β is a protein encoded by 1533 bp cDNA having the basesequence of SEQ ID NO: 3, and this has the sequence of 510 amino acidsas in SEQ ID NO: 4.

[0026] Nectin-3γ is a protein encoded by 1317 bp cDNA having the basesequence of SEQ ID NO: 5, and this has the sequence of 435 amino acidsas in SEQ ID NO: 6.

[0027] These proteins nectin-3 can be obtained in any known method, forexample, according to a method of isolating them from mouse and othermammal tissues; according to a method that comprises preparing peptidesthrough chemical synthesis based on the amino acid sequences provided bythis invention; or according to a method of recombinant DNA technologyusing the polynucleotides provided by this invention. Concretely,nectin-3 may be obtained through recombinant DNA technology as follows:An RNA is prepared through in-vitro transcription from a vector having apolynucleotide that encodes nectin-3, and the nectin-3 is expressed invitro through in vitro translation using the RNA as a template. In thecase of the polynucleotide encoding the nectin-3 being recombined with asuitable expression vector in a known manner, the nectin-3 encoded bythe polynucleotide can be abundantly expressed in E. coli, B. subtilis,yeast, animal or plant cells, etc.

[0028] The protein nectin-3 of this invention can be expressed inmicroorganisms such as E. coli, in the following manner: Thepolynucleotide of this invention that encodes the protein is insertedinto an expression vector having an origin replicavable inmicroorganisms, a promoter, a ribosome-binding site, a DNA cloning siteand a terminator to construct a recombinant expression vector; then hosecells are transformed with the expression vector; and the resultingtransformant cells are cultured. In that manner, the nectin-3 encoded bythe polynucleotide can be abundantly produced in microorganisms.Alternatively, it may be expressed in the form of a fusion protein withany other protein segment. The resulting fusion protein is cleaved witha suitable protease, and only the protein segment encoded by thepolynucleotide can be obtained.

[0029] The protein nectin-3 of this invention can be expressed in animalcells in the following manner: The polynucleotide of this invention thatencodes the protein is recombined with an expression vector for animalcells having a promoter, a splicing region and a poly(A)-addition site,and the recombinant vector is introduced into animal cells, whereby theprotein nectin-3 of this invention can be expressed in thethus-transformed animal cells.

[0030] The mouse protein nectin-3 obtained according to the method asabove can be used, for example, as an antigen to form an antibody thatspecifically recognizes this protein.

[0031] The protein nectin-3 includes peptide fragments (of at lease 5amino acid residues) having any partial amino acid sequence of the aminoacid sequence of SEQ ID NO: 2, 4 or 6. These peptide fragments are alsousable as an antigen for constructing the antibody.

[0032] The polynucleotide of this invention is a genomic gene of mammalsthat encodes any of the above-mentioned protein nectin-3α, β or γ. Forexample, it can be isolated from known genomic libraries, using thepolynucleotide having the base sequence of any SEQ ID NO: 1, 3 or 5 orits partial sequence as a probe.

[0033] A polynucleotide of this invention may be cDNA characterized byhaving the base sequence of SEQ ID NO: 1, 3 or 5, and it encodes any ofthe above-mentioned nectin-3α, β or γ. Clones of the polynucleotides ofthis invention can be obtained with ease. Concretely, using anoligonucleotide probe synthesized on the bases of the base sequence ofSEQ ID NO: 1, 3 or 5, mouse and other mammal cDNA libraries arescreened. Alternatively, using the oligonucleotides as primers, theintended polynucleotides can be synthesized through polymerase chainreaction (PCR).

[0034] In general, mammal genes include many polymorphs owing toindividual differences. Therefore, polynucleotides modified from thosehaving a base sequence of any of SEQ ID NO: 1, 3 or 5 by adding,deleting and/or substituting one or more nucleotides therein with anynucleotide(s) are all within the scope of this invention. Similarly,proteins modified through the polynucleotide modification to add, deleteand/or substitute one or more amino acid residues with any other aminoacid residue(s) are also all within the scope of this invention, so faras they have the activity of the protein having the amino acid sequenceof any of SEQ ID NO: 2, 4 or 6.

[0035] The polynucleotide of this invention includes DNA fragments (ofat least 10 bp) having any partial base sequence of the base sequence ofSEQ ID NO: 1, 3 or 5, and also polynucleotides comprising theirantisense chains.

[0036] The antibody of this invention can be obtained as a polyclonalantibody or a monoclonal antibody in any known method of using theprotein nectin-3 itself or its partial peptide as an antigen.

[0037] The following Examples are to show the experimental data toconfirm the structure and the function of the nectin-3 of thisinvention.

EXAMPLES

[0038] 1. Procedures

[0039] 1.1 Molecular Cloning of Mouse Nectin-3 cDNAs

[0040] From the EST database, three different types of mouse EST clones(AI1428160, AA492633, AA497887), which are similar to but are not thesame as nectin-1 and -2, were amplified from a mouse brain cDNA(Clontech). Using the mixture of these cDNAs as a probe, a mouse cDNAlibrary (Stratagene) was screened to obtain a full-length cDNA. For isDNA sequencing, used was a DNA sequencer (ABI 373).

[0041] 1.2 Construction of Nectin-3 Expression Vectors

[0042] Vectors pCAGIPuro (J. Biol. Chem., 275:613-618, 2000),pCAGIPuro-FLAG, p-GEX-KG (Anal. Biochem., 192: 262-267, 1991), pMal-C2(New England Biolabs Inc.) and pFastBAc1-Msp-Fc (J. Cell. biol., 145:539-549, 1999) were used for constructing nectin-3 expression vectorsmentioned below. The pCAGIPuro-FLAG was constructed by subcloning aprepro-trypsin signal peptide and the FLAG epitope of p FLAG-CMV1(Eastman Kodak) into pCAGIPuro.

[0043] (a) pCAGIPuro-nectin-3α: 1-549 amino acids (full length) of SEQID NO: 2,

[0044] (b) pCAGIPuro-FLAG-nectin-3α: 56-549 amino acids of SEQ ID NO: 2,

[0045] (c) GST-nectin-3α-CP: 433-549 amino acids of SEQ ID NO: 2(cytoplasmic region),

[0046] (d) GST-nectin-3α-CP-ΔC: 433-545 amino acids of SEQ ID NO: 2(deletion of the C-terminal four amino acid residues),

[0047] (e) GST-nectin-3γ^ CP: 397-438 amino acids of SEQ ID NO: 6(cytoplasmic region),

[0048] (f) pFastBac1-Msp-Fc-nectin-3α-EX: 56-400 amino acids of SEQ IDNO: 2 (extracellular region)

[0049] 1.3 Construction of Transformant Cells

[0050] L cells (obtained from the Kyoto University) were cultured in a10% fetal calf serum-containing DMEM medium to prepare parental L cellsfor transformation. Full-length human nectin-1α-expressing L cells(nectin-1α-L cells) and full-length mouse nectin-2α-expressing L cells(nectin-2α-L cells) were constructed according to the method describedin references (J. Cell Biol., 145: 539-549, 1999; J. Biol. Chem., 275:613-618, 2000). Full-length mouse nectin-3α-expressing L cells(nectin-3α-L cells) were constructed, using a recombinant vectorpCAGIPuro-nectin-3α. Nectin-1α and FLAG-nectin-3α-coexpressing L cells(nectin-1α/3α-L cells), and nectin-2α and FLAG-nectin-3α-coexpressing Lcells (nectin-2α/3α-L cells) were constructed by transfecting thenectin-1α-L cells and the nectin-2α-L cells, respectively, withpCAGIPuro-FLAG-nectin-3α, using a lipofectamine reagent (GIBCO BRL).Each cell line was cultured for 1 day, replated, and selected byculturing in the present of 5 μg/ml of puromycin (Sigma Chemical Co.).

[0051] 1.4 Preparation of Antibodies

[0052] A rabbit antiserum (polyclonal antibody) against nectin-3α wasraised against an antigen, GST-nectin-3α-CP. A rat monoclonal antibodyagainst nectin-3 was raised against an antigen, fusion protein of theextracellular region of nectin-3α with IgG Fc. A rabbit polyclonalanti-nectin-1α antibody was prepared according to the method describedin a reference (J. Cell Biol., 145: 539-549, 1999). Another rabbitpolyclonal anti-nectin-1α antibody was raised against an antigen,synthetic peptide corresponding to the amino acid sequence 450-468 ofnectin-1α, according to the method described in the reference (J. CellBiol., 145: 539-549, 1999). A rat monoclonal anti-nectin-2 antibody wasprepared according to the method described in references (J. Cell Biol.,145: 539-549, 1999; Exp. Cell Res., 235: 374-384, 1997); and mousemonoclonal and rabbit polyclonal anti-1-afadin antibodies were preparedaccording to the method described in references (J. Cell Biol., 139:517-528, 1997; Oncogene 18: 1609-1618, 1999). A rat monoclonalanti-E-cadherin antibody was obtained from Dr. Takeichi (KyotoUniversity). A mouse monoclonal anti-FLAG antibody was bought fromEastman Kodak.

[0053] 1.5 Other Procedures

[0054] Cell aggregation assay was done according to the method describedin references (J. Cell Biol., 145: 539-549, 1999; J. Biol. Chem., 275;613-618, 2000). For mixed-cell aggregation assay between two different Lcell lines, one L cell line was prelabeled with DiI (Molecular ProbeInc., USA), as described (J. Cell Biol., 103: 171-187, 1986).

[0055] Chemical cross-linking was done according to the method describedin references (Blood 92: 4602-4611, 1998; J. Biol. Chem., 275: 613-618,2000). Immunoprecipitation was performed according to the methoddescribed in references (J. Cell Biol., 145: 539-549, 1999; J. Biol.Chem., 275: 613-618, 2000). Immunofluorescence microscopy of culturedcells was done according to the method described in references (J. CellBiol., 139: 517-528, J. Cell Biol., 145: 539-549, 1999; J. Biol. Chem.,275: 613-618, 2000). Protein concentrations were determined with bovineserum albumin as a control, according to the method described in areference (Anal. Biochem., 72: 248-254, 1976). SDS-PAGE was done asdescribed (Nature, 227: 680-685, 1970).

[0056] Affinity chromatography was performed as follows: The MBP-fusionprotein of the PDZ domain of afadin was immobilized on amylose resinbeads (New England Biolabs Inc.) GST-nectin-3α-CP andGST-nectin-3α-CP-ΔC were separately applied to the affinity beads. Afterextensively washed with PBS (containing 0.1% Triton X-100), the beadswere subjected to elution with PBS (containing 20 mM maltose, 0.1%Triton X-100).

[0057] 2. Results

[0058] 2.1 Cloning and Characterization of Nectin-3 cDNAs

[0059] The cDNA clone obtained from the mouse cDNA library has the basesequence of SEQ ID NO: 7, and encoded a protein (calculated molecularweight: 60,580) comprising 549 amino acids (SEQ ID NO: 2) in the 1647 bpcoding regions (SEQ ID NO: 1). This clone contained all theabove-mentioned EST clones. The protein was named nectin-3α.

[0060] The amino acid sequence of The nectin-3α had an N-terminalhydrophobic signal peptide (1-55 amino acids of SEQ ID NO: 2) and atransmembrane region (405-421 amino acids of SEQ ID NO: 2). The sitesfor N-linked glycosylation were detected at 73, 83, 125, 186, 222 and331 amino acids. The nectin-3 contained three lg-like domains in theextracellular region and a C-terminal conserved motif in the cytoplasmicregion (Table 1). TABLE 1 C-Terminal Sequences of the Nectin FamilyMembers Nectin-1α SFISKKEWYV Nectin-1β VRTTEPRGEC Nectin-2α SLISRRAVYVNectin-2δ DEFVSRAMYV Nectin-3α SVISRREWYV Nectin-3β LYINPREHYV Nectin-3γLGQVRALEDT

[0061] As in the above, these structural properties of nectin-3α aresimilar to those of nectin-1α, -1β, -2α and -2δ. The extent of homologyvaried with the regions of the molecules, but the amino acid sequence ofthe extracellular region of nectin-3α showed 35.9% and 30.7% identitiesto those of nectin-1 and nectin-2, respectively.

[0062] During the isolation of nectin-3α, two splicing variants(nectin-3β and -3γ) were isolated. The nectin-3β cDNA (coding region)has a base sequence of 1533 bps (SEQ ID NO: 3), and encodes a proteinhaving an amino acid sequence of 510 amino acids (SEQ ID NO: 4,calculated molecular weight: 55,808). The extracellular region ofnectin-3β (1-357 amino acids of SEQ ID NO: 4) is identical to that ofnectin-3α, but its transmembrance and cytoplasmic regions (358-510 aminoacids of SEQ ID NO: 4) are different from those of nectin-3α. However,nectin-3β also has a C-terminal conserved motif (Table 1).

[0063] The nectin-3γ cDNA (coding region) has a base sequence of 1317bps (SEQ ID NO: 5), and encodes a protein having an amino acid sequenceof 438 amino acids (SEQ ID NO: 6, calculated molecular weight: 47,259).The extracellular region, transmembrane region and cytoplasmic region ofnectin-3γ are identical to those of nectin-3β, but nectin-3γ lacks theC-terminal conserved motif (Table 1).

[0064]FIG. 1 is to compare the amino acid sequences (in one-lettercoding) of these nectin-3α, -3β and -3γ.

[0065] The following experiments are principally directed to nectin-3α.This is because the data in Northern blot analysis of various tissuesconfirm that nectin-3α is a major splicing variant (see FIG. 8, B1 toB3).

[0066] 2.2 trans Homo-interaction and cis Homo-dimer Formation

[0067] Studies with nectin-1α-L cells and nectin-2α-L cells confirm thatnectin-1α and nectin-2α show cell-cell adhesion activity (transhomo-interaction) (J. Cell Biol., 145: 539-549, 1999; J. Biol. Chem.,275: 613-618, 2000). With that, here in examined was whether nectin-3αalso shows the same activity. The polyclonal anti-nectin-3α antibodyrecognized two protein bands with molecular masses of about 100 kDA inthe expression products by nectin-3α-L cells (FIG. 2A, FIG. 3). This maybe dues to the different levels of the post-translational modificationsuch as glycosylation. These molecular masses are different from thecalculated molecular weight based on the deduced amino acid sequence.This difference may also be due to the glycosylation. The expressionlevel of nectin-1α, -2α and -3α in each cell line was almost the same(data not shown).

[0068] Nectin-3α-L cells were tested for the cell aggregation activityof nectin-3α. As a result, the cell aggregation activity of nectin-3αvaried time-dependently (FIG. 2B, C1, C2). EDTA added to nectin-3α didnot have any influence on this activity of nectin-3α (data not shown).This confirms that the cell-cell adhesion activity of nectin-3α does notdepend on Ca²⁺. These results indicate that nectin-3α is aCa²⁺-independent homophilic CAM (cell adhesion molecule), likenectin-1α, -2α and -2δ (J. Cell Biol., 145: 539-549, 1999; Exp. CellRes., 235: 374-384, 1997; Blood, 92: 4602-4611, 1998; J. Biol. Chem.,275: 613-618, 2000).

[0069] Nectin-1α and -2α are known to show cis homo-dimerization (J.Biol. Chem., 275: 613-618, 2000). With that, nectin-3α was also testedfor the same activity. Nectin-3α-L cells were dissociated to asingle-cell suspension and incubated with a cell surface cross-linker,BS3, and then subjected to Western blot analysis using a polyclonalanti-nectin-3α antibody. The cross-linking of the cell line resulted inthe formation of additional bands with molecular masses of about 200 to220 kDa that correspond to dimers (FIG. 3). Bands with higher molecularmasses were also detected. Because the cross-linking was done in asingle-cell suspension, it is most likely that the dimers and oligomersare derived from the cis homo-interaction rather that from the transhomo-interaction.

[0070] 2.3 trans Hetero-interaction of Nectin-3α with Nectin-1α or -2α

[0071] To examine whether each member of the nectin family showsheterophilic cell-cell adhesion activity (trans hetero-interaction),mixed-cell aggregation assay was performed. When DiI-labeled nectin-1α-Lcells were mixed with unlabeled nectin-2α-L cells, the resultingaggregates exclusively consisted of the labeled cells alone or theunlabeled cells alone (FIG. 4 A1-A3). Few aggregates consisting of boththe labeled and unlabeled cells were detected. In contrast, whenDiI-labeled nectin-3α-L cells were mixed with unlabeled nectin-1α-Lcells, the resulting aggregates consisted of both the labeled cells andthe unlabeled cells (FIG. 4, B1-B3). This indicates that nectin-3α showstrans hetero-interaction with nectin-1α. The similar result was obtainedwith nectin-3α and nectin-2α (FIG. 4, C1-C3).

[0072] To confirm these results, immunofluorescence microscopy wasperformed. When nectin-1α-L cells were co-cultured with nectin-2α-Lcells, nectin-1α and nectin-2α were localized in the cell-cell contactsites of the respective L cells (FIG. 5, A1-A3). However, neithernectin-1α or nectin-2α was detected in the contact sites of the twotypes of the L cells. In contrast, when nectin-3α-L cells wereco-cultured with nectin-1α-L cells, nectin-3α and nectin-1α coexisted inthe cell-cell contact site of the two types of the L cells (FIG. 5,B1-B3). The same result was obtained in co-culture of nectin-3α-L cellsand nectin-2α-L cells (FIG. 5, C1-C3). These result indicate thatnectin-3α shows trans hetero-interaction with nectin-1α and -2α, whereasnectin-1α does not trans hetero-interaction with nectin-2α.

[0073] To determine whether each member of the nectin family preferstrans homo-interaction or trans hetero-interaction, two-cell aggregateswere analyzed through mixed-cell aggregation assay. Consistent with theresult in the analysis of two-cell aggregates, the mixture ofnectin-1α-L cells and nectin-2α-L cells resulted in the formation ofhomotypic two-cell aggregates (FIG. 6A). In contrast, the mixture ofnectin-1α-L cells and nectin-3α-L cells resulted in the formation ofheterotypic two-cell aggregates, and few homotypic two-cell aggregateswere found (FIG. 6B). The similar result was obtained from nectin-3α-Lcells and nectin-2α-L cells were mixed (FIG. 6C). These results indicatethat the affinity of trans hetero-interaction of nectin-3α withnectin-1α or -2α is obviously higher than that of trans homo-interactionof nectin-1α, -2α or -3α.

[0074] 2.4 cis Hetero-dimerization of Nectin-3α with Nectin-1α or -2α

[0075] Next examined was whether nectin-3α forms a cis hetero-dimer withnectin-1α or -2α. Using nectin-1α/3α-L cells and nectin-2α/3α-L cells,FLAG-nectin-3α was expressed in the nectin-1α-L cells and thenectin-2α-L cells. As a result, the FLAG-nectin-3α expressed did notchange the size of the cis dimers of nectin-1α or -2α (FIG. 7, A1 andA2). When nectin-1α/3α-L cells were subjected to cell surfacecross-linking, followed by immunoprecipitation using the monoclonalanti-FLAG antibody, then nectin-1α was recovered in the supernatant andwas not coimmunoprecipitated with FLAG-nectin-3α (FIG. 7, B1). thesimilar result was obtained with nectin-2α/3α-L cells (FIG. 7, B2).These results indicate that nectin-3α does not form a cis hetero-dimerwith nectin-1α or -2α.

[0076] 2.5 Tissue Distribution and Intracellular Localization ofNectin-3α

[0077] Consistent with previous reports (J. Virol., 66: 2807-2813, 1992;Gene 155: 261-265, 1995; Gene 159: 267-272, 1995), Northern blotanalysis revealed that nectin-1 is abundantly expressed in brain whereasnectin-2 is ubiquitously expressed (FIG. 8, A1 and A2). Northern blotanalysis using, as a probe, the coding region common to the threesplicing variants of nectin-3 revealed some mRNA bands in varioustissues (FIG. 8, A3). To determine the tissue distribution of eachsplicing variant, a cDNA probe specific to each variant was used.Nectin-3α gave about 5.2-kb, 3.8-kb, 3.3-kb and 2.7-kb mRNA bands,abundantly expressed in testis but slightly in other tissues (heart,brain, lung, liver and kidney) (FIG. 8, B1). Nectin-3β gave about 5.2-kband 3.3-kb mRNA bands, expressed in testis (FIG. 8, B2). Nectin-3γ gavean about 3.3-kb mRNA band in testis, and an about 2.1-kb mRNA bank inlung, liver and kidney (FIG. 8, B3).

[0078] To determine the intracellular localization of nectin-3α,immunofluorescence microscopy was performed, using a polyclonalanti-nectin-3α antibody. Nectin-3α was colocalized with nectin-2 in thejunctional complex regions in mouse small intestine absorptive epithelia(FIG. 9). These results suggest that nectin-3α is also localized atcadherin-based cell-cell AJs, like nectin-2 (J. Cell Biol., 145:539-549, 1999).

[0079] 2.6 Direct Binding of Nectin-3 to Afadin

[0080] to confirm whether nectin-3α directly binds to afadin, affinitychromotography was performed. The GST-fusion protein in the cytoplasmicregion of nectin-3α (GST-nectin-3α-CP) bound to the MBP-fusion proteinin the PDZ domain of afadin )MBP-afadin-PDZ) immobilized on amyloseresin beads (FIG. 10). The stoichiometry of binding of nectin-3α toafadin was about 1:1. In contrast, the GST-fusion protein in theC-terminal four amino acid residues-deleted cytoplasmic region ofnectin-3α (GST-nectin-3α-CP-ΔC) did not bind to the affinity beads.Similarly, the GST-fusion protein in the cytoplasmic region of nectin-3γthat lacks the C-terminal conserved motif did not also bind to theaffinity beads.

INDUSTRIAL APPLICABILITY

[0081] As described in detail hereinabove, the invention of thisapplication provides a novel protein nectin-3 that belongs to one andthe same protein family to which nectin-1 and -2 belong. The proteinprovides important information for clarifying all aspects of themolecular mechanism in cell-cell binding systems, and, in addition, itleads to the possibility of clarifying the mechanism of, for example,humectation and metastasis of carcinoma, and is expected to beapplicable to diagnosis of carcinoma for its malignancy and to a methodfor treating cases with carcinoma and also to development of medicinesfor carcinoma.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 14 <210> SEQ ID NO 1<211> LENGTH: 1650 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1650) <300> PUBLICATIONINFORMATION: <301> AUTHORS: Satoh-Horikawa K. et al. <302> TITLE:Nectin-3, a new member of immunoglobulin-like cell adhesion moleculesthat shows homophilic and heterophilic cell-cell adhesion activities.<303> JOURNAL: J. Biol. Chem. <304> VOLUME: 275 <305> ISSUE: 14 <306>PAGES: 10291-10299 <307> DATE: 2000-04-07 <400> SEQUENCE: 1 atg gcg cggacc ccg ggc ccg gcc ccg ttg tgt cct gga ggc ggc aaa 48 Met Ala Arg ThrPro Gly Pro Ala Pro Leu Cys Pro Gly Gly Gly Lys 1 5 10 15 gca caa ctttcc tcg gcg ttt cct ccc gcg gcc gga ctg ctg ctg ccg 96 Ala Gln Leu SerSer Ala Phe Pro Pro Ala Ala Gly Leu Leu Leu Pro 20 25 30 gcc ccg acg ccgccg ccg ctg ctg ctg ctg ctt att ccc ctg ctt ctc 144 Ala Pro Thr Pro ProPro Leu Leu Leu Leu Leu Ile Pro Leu Leu Leu 35 40 45 ttc tcc cgg ctc tgtggt gcc tta gct gga tca att att gtg gag cca 192 Phe Ser Arg Leu Cys GlyAla Leu Ala Gly Ser Ile Ile Val Glu Pro 50 55 60 cat gtc aca gca gtg tgggga aag aat gtt tca ttg aag tgt tta att 240 His Val Thr Ala Val Trp GlyLys Asn Val Ser Leu Lys Cys Leu Ile 65 70 75 80 gaa gtg aat gaa act ataacc cag atc tca tgg gag aag ata cat ggc 288 Glu Val Asn Glu Thr Ile ThrGln Ile Ser Trp Glu Lys Ile His Gly 85 90 95 aaa agt aca cag act gtt gcagtt cat cat cct cag tat gga ttc tct 336 Lys Ser Thr Gln Thr Val Ala ValHis His Pro Gln Tyr Gly Phe Ser 100 105 110 gtt caa gga gat tat cag ggaaga gtc ttg ttt aaa aac tat tca ctt 384 Val Gln Gly Asp Tyr Gln Gly ArgVal Leu Phe Lys Asn Tyr Ser Leu 115 120 125 aat gat gca aca att act ctgcat aac ata ggc ttc tca gat tct gga 432 Asn Asp Ala Thr Ile Thr Leu HisAsn Ile Gly Phe Ser Asp Ser Gly 130 135 140 aaa tat ata tgc aaa gcc gttaca ttc cca ctt gga aat gct cag tcc 480 Lys Tyr Ile Cys Lys Ala Val ThrPhe Pro Leu Gly Asn Ala Gln Ser 145 150 155 160 tct aca aca gtg act gtgtta gtt gaa ccc aca gtg agc ctg ata aaa 528 Ser Thr Thr Val Thr Val LeuVal Glu Pro Thr Val Ser Leu Ile Lys 165 170 175 ggg ccg gat tct tta attgat gga ggg aat gag aca gta gca gcc gtt 576 Gly Pro Asp Ser Leu Ile AspGly Gly Asn Glu Thr Val Ala Ala Val 180 185 190 tgt gta gca gcc act ggaaag cca gtc gca cag att gac tgg gaa ggt 624 Cys Val Ala Ala Thr Gly LysPro Val Ala Gln Ile Asp Trp Glu Gly 195 200 205 gat ctt ggt gaa atg gaatct agt aca act tct ttt cct aat gaa aca 672 Asp Leu Gly Glu Met Glu SerSer Thr Thr Ser Phe Pro Asn Glu Thr 210 215 220 gca acg att gtc agc caatac aag ctg ttt ccc aca aga ttt gct cga 720 Ala Thr Ile Val Ser Gln TyrLys Leu Phe Pro Thr Arg Phe Ala Arg 225 230 235 240 gga agg cga att acttgt gtt gta aaa cat cca gcc tta gaa aag gac 768 Gly Arg Arg Ile Thr CysVal Val Lys His Pro Ala Leu Glu Lys Asp 245 250 255 att cgc tac tct ttcata cta gac ata cag tat gct cct gaa gtt tca 816 Ile Arg Tyr Ser Phe IleLeu Asp Ile Gln Tyr Ala Pro Glu Val Ser 260 265 270 gta aca gga tat gatgga aat tgg ttc gtg gga aga aaa ggt gtt aac 864 Val Thr Gly Tyr Asp GlyAsn Trp Phe Val Gly Arg Lys Gly Val Asn 275 280 285 ctc aag tgt aat gctgat gca aac cct cca ccc ttc aag tcc gtg tgg 912 Leu Lys Cys Asn Ala AspAla Asn Pro Pro Pro Phe Lys Ser Val Trp 290 295 300 agc agg ttg gat ggacaa tgg cct gat ggt tta ttg gcg tca gat aat 960 Ser Arg Leu Asp Gly GlnTrp Pro Asp Gly Leu Leu Ala Ser Asp Asn 305 310 315 320 act ctt cat tttgtc cat cca ttg act gtc aat tat tct ggc gtt tat 1008 Thr Leu His Phe ValHis Pro Leu Thr Val Asn Tyr Ser Gly Val Tyr 325 330 335 gtc tgt aaa gtatca aat tcc ctt ggt caa aga agt gat caa aag gtt 1056 Val Cys Lys Val SerAsn Ser Leu Gly Gln Arg Ser Asp Gln Lys Val 340 345 350 atc tac att tcagat cct cct acc acc acc acc ctt cag ccg aca gtt 1104 Ile Tyr Ile Ser AspPro Pro Thr Thr Thr Thr Leu Gln Pro Thr Val 355 360 365 cag tgg cat tcctca cct gct gac gtc cag gat ata gca aca gag cat 1152 Gln Trp His Ser SerPro Ala Asp Val Gln Asp Ile Ala Thr Glu His 370 375 380 aaa aaa ttg cccttt cct ttg tca act ttg gca aca ctt aag gat gac 1200 Lys Lys Leu Pro PhePro Leu Ser Thr Leu Ala Thr Leu Lys Asp Asp 385 390 395 400 aca att ggcacc atc att gct agt gta gtg ggt ggg gct ctc ttc tta 1248 Thr Ile Gly ThrIle Ile Ala Ser Val Val Gly Gly Ala Leu Phe Leu 405 410 415 gtg ctt gtgagc att tta gct ggg gta ttc tgc tat agg aga cga cgg 1296 Val Leu Val SerIle Leu Ala Gly Val Phe Cys Tyr Arg Arg Arg Arg 420 425 430 acg ttt cgtgga gac tac ttt gcc aaa aac tac att cca cca tca gac 1344 Thr Phe Arg GlyAsp Tyr Phe Ala Lys Asn Tyr Ile Pro Pro Ser Asp 435 440 445 atg cag aaagaa tca cag att gat gtt ctt cac cag gat gag ctg gat 1392 Met Gln Lys GluSer Gln Ile Asp Val Leu His Gln Asp Glu Leu Asp 450 455 460 tct tac ccagac agt gta aaa aag gaa aac aaa aat cca gta aac aac 1440 Ser Tyr Pro AspSer Val Lys Lys Glu Asn Lys Asn Pro Val Asn Asn 465 470 475 480 ctg atccgc aaa gac tac tta gag gag cct gag aaa act cag tgg aat 1488 Leu Ile ArgLys Asp Tyr Leu Glu Glu Pro Glu Lys Thr Gln Trp Asn 485 490 495 aat gtagag aac ctc act agg ttt gaa aga ccg atg gat tac tat gaa 1536 Asn Val GluAsn Leu Thr Arg Phe Glu Arg Pro Met Asp Tyr Tyr Glu 500 505 510 gat ctaaaa atg gga atg aag ttt gtc agt gat gaa cgc tac aat gaa 1584 Asp Leu LysMet Gly Met Lys Phe Val Ser Asp Glu Arg Tyr Asn Glu 515 520 525 agt gaagat ggt ttg gtt tct cat gta gat ggc tcc gta att tcc agg 1632 Ser Glu AspGly Leu Val Ser His Val Asp Gly Ser Val Ile Ser Arg 530 535 540 agg gagtgg tat gtc taa 1650 Arg Glu Trp Tyr Val 545 <210> SEQ ID NO 2 <211>LENGTH: 549 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 2 MetAla Arg Thr Pro Gly Pro Ala Pro Leu Cys Pro Gly Gly Gly Lys 1 5 10 15Ala Gln Leu Ser Ser Ala Phe Pro Pro Ala Ala Gly Leu Leu Leu Pro 20 25 30Ala Pro Thr Pro Pro Pro Leu Leu Leu Leu Leu Ile Pro Leu Leu Leu 35 40 45Phe Ser Arg Leu Cys Gly Ala Leu Ala Gly Ser Ile Ile Val Glu Pro 50 55 60His Val Thr Ala Val Trp Gly Lys Asn Val Ser Leu Lys Cys Leu Ile 65 70 7580 Glu Val Asn Glu Thr Ile Thr Gln Ile Ser Trp Glu Lys Ile His Gly 85 9095 Lys Ser Thr Gln Thr Val Ala Val His His Pro Gln Tyr Gly Phe Ser 100105 110 Val Gln Gly Asp Tyr Gln Gly Arg Val Leu Phe Lys Asn Tyr Ser Leu115 120 125 Asn Asp Ala Thr Ile Thr Leu His Asn Ile Gly Phe Ser Asp SerGly 130 135 140 Lys Tyr Ile Cys Lys Ala Val Thr Phe Pro Leu Gly Asn AlaGln Ser 145 150 155 160 Ser Thr Thr Val Thr Val Leu Val Glu Pro Thr ValSer Leu Ile Lys 165 170 175 Gly Pro Asp Ser Leu Ile Asp Gly Gly Asn GluThr Val Ala Ala Val 180 185 190 Cys Val Ala Ala Thr Gly Lys Pro Val AlaGln Ile Asp Trp Glu Gly 195 200 205 Asp Leu Gly Glu Met Glu Ser Ser ThrThr Ser Phe Pro Asn Glu Thr 210 215 220 Ala Thr Ile Val Ser Gln Tyr LysLeu Phe Pro Thr Arg Phe Ala Arg 225 230 235 240 Gly Arg Arg Ile Thr CysVal Val Lys His Pro Ala Leu Glu Lys Asp 245 250 255 Ile Arg Tyr Ser PheIle Leu Asp Ile Gln Tyr Ala Pro Glu Val Ser 260 265 270 Val Thr Gly TyrAsp Gly Asn Trp Phe Val Gly Arg Lys Gly Val Asn 275 280 285 Leu Lys CysAsn Ala Asp Ala Asn Pro Pro Pro Phe Lys Ser Val Trp 290 295 300 Ser ArgLeu Asp Gly Gln Trp Pro Asp Gly Leu Leu Ala Ser Asp Asn 305 310 315 320Thr Leu His Phe Val His Pro Leu Thr Val Asn Tyr Ser Gly Val Tyr 325 330335 Val Cys Lys Val Ser Asn Ser Leu Gly Gln Arg Ser Asp Gln Lys Val 340345 350 Ile Tyr Ile Ser Asp Pro Pro Thr Thr Thr Thr Leu Gln Pro Thr Val355 360 365 Gln Trp His Ser Ser Pro Ala Asp Val Gln Asp Ile Ala Thr GluHis 370 375 380 Lys Lys Leu Pro Phe Pro Leu Ser Thr Leu Ala Thr Leu LysAsp Asp 385 390 395 400 Thr Ile Gly Thr Ile Ile Ala Ser Val Val Gly GlyAla Leu Phe Leu 405 410 415 Val Leu Val Ser Ile Leu Ala Gly Val Phe CysTyr Arg Arg Arg Arg 420 425 430 Thr Phe Arg Gly Asp Tyr Phe Ala Lys AsnTyr Ile Pro Pro Ser Asp 435 440 445 Met Gln Lys Glu Ser Gln Ile Asp ValLeu His Gln Asp Glu Leu Asp 450 455 460 Ser Tyr Pro Asp Ser Val Lys LysGlu Asn Lys Asn Pro Val Asn Asn 465 470 475 480 Leu Ile Arg Lys Asp TyrLeu Glu Glu Pro Glu Lys Thr Gln Trp Asn 485 490 495 Asn Val Glu Asn LeuThr Arg Phe Glu Arg Pro Met Asp Tyr Tyr Glu 500 505 510 Asp Leu Lys MetGly Met Lys Phe Val Ser Asp Glu Arg Tyr Asn Glu 515 520 525 Ser Glu AspGly Leu Val Ser His Val Asp Gly Ser Val Ile Ser Arg 530 535 540 Arg GluTrp Tyr Val 545 <210> SEQ ID NO 3 <211> LENGTH: 1533 <212> TYPE: DNA<213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(1533) <300> PUBLICATION INFORMATION: <301> AUTHORS: Satoh-HorikawaK. et al. <302> TITLE: Nectin-3, a new member of immunoglobulin-likecell adhesion molecules that shows homophilic and heterophilic cell-celladhesion activities. <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 275<305> ISSUE: 14 <306> PAGES: 10291-10299 <307> DATE: 2000-04-07 <308>DATABASE ACCESSION NUMBER: GenBank accession No. AF195834 <309> DATABASEENTRY DATE: 2000-04-13 <400> SEQUENCE: 3 atg gcg cgg acc ccg ggc ccg gccccg ttg tgt cct gga ggc ggc aaa 48 Met Ala Arg Thr Pro Gly Pro Ala ProLeu Cys Pro Gly Gly Gly Lys 1 5 10 15 gca caa ctt tcc tcg gcg ttt cctccc gcg gcc gga ctg ctg ctg ccg 96 Ala Gln Leu Ser Ser Ala Phe Pro ProAla Ala Gly Leu Leu Leu Pro 20 25 30 gcc ccg acg ccg ccg ccg ctg ctg ctgctg ctt att ccc ctg ctt ctc 144 Ala Pro Thr Pro Pro Pro Leu Leu Leu LeuLeu Ile Pro Leu Leu Leu 35 40 45 ttc tcc cgg ctc tgt ggt gcc tta gct ggatca att att gtg gag cca 192 Phe Ser Arg Leu Cys Gly Ala Leu Ala Gly SerIle Ile Val Glu Pro 50 55 60 cat gtc aca gca gtg tgg gga aag aat gtt tcattg aag tgt tta att 240 His Val Thr Ala Val Trp Gly Lys Asn Val Ser LeuLys Cys Leu Ile 65 70 75 80 gaa gtg aat gaa act ata acc cag atc tca tgggag aag ata cat ggc 288 Glu Val Asn Glu Thr Ile Thr Gln Ile Ser Trp GluLys Ile His Gly 85 90 95 aaa agt aca cag act gtt gca gtt cat cat cct cagtat gga ttc tct 336 Lys Ser Thr Gln Thr Val Ala Val His His Pro Gln TyrGly Phe Ser 100 105 110 gtt caa gga gat tat cag gga aga gtc ttg ttt aaaaac tat tca ctt 384 Val Gln Gly Asp Tyr Gln Gly Arg Val Leu Phe Lys AsnTyr Ser Leu 115 120 125 aat gat gca aca att act ctg cat aac ata ggc ttctca gat tct gga 432 Asn Asp Ala Thr Ile Thr Leu His Asn Ile Gly Phe SerAsp Ser Gly 130 135 140 aaa tat ata tgc aaa gcc gtt aca ttc cca ctt ggaaat gct cag tcc 480 Lys Tyr Ile Cys Lys Ala Val Thr Phe Pro Leu Gly AsnAla Gln Ser 145 150 155 160 tct aca aca gtg act gtg tta gtt gaa ccc acagtg agc ctg ata aaa 528 Ser Thr Thr Val Thr Val Leu Val Glu Pro Thr ValSer Leu Ile Lys 165 170 175 ggg ccg gat tct tta att gat gga ggg aat gagaca gta gca gcc gtt 576 Gly Pro Asp Ser Leu Ile Asp Gly Gly Asn Glu ThrVal Ala Ala Val 180 185 190 tgt gta gca gcc act gga aag cca gtc gca cagatt gac tgg gaa ggt 624 Cys Val Ala Ala Thr Gly Lys Pro Val Ala Gln IleAsp Trp Glu Gly 195 200 205 gat ctt ggt gaa atg gaa tct agt aca act tctttt cct aat gaa aca 672 Asp Leu Gly Glu Met Glu Ser Ser Thr Thr Ser PhePro Asn Glu Thr 210 215 220 gca acg att gtc agc caa tac aag ctg ttt cccaca aga ttt gct cga 720 Ala Thr Ile Val Ser Gln Tyr Lys Leu Phe Pro ThrArg Phe Ala Arg 225 230 235 240 gga agg cga att act tgt gtt gta aaa catcca gcc tta gaa aag gac 768 Gly Arg Arg Ile Thr Cys Val Val Lys His ProAla Leu Glu Lys Asp 245 250 255 att cgc tac tct ttc ata cta gac ata cagtat gct cct gaa gtt tca 816 Ile Arg Tyr Ser Phe Ile Leu Asp Ile Gln TyrAla Pro Glu Val Ser 260 265 270 gta aca gga tat gat gga aat tgg ttc gtggga aga aaa ggt gtt aac 864 Val Thr Gly Tyr Asp Gly Asn Trp Phe Val GlyArg Lys Gly Val Asn 275 280 285 ctc aag tgt aat gct gat gca aac cct ccaccc ttc aag tcc gtg tgg 912 Leu Lys Cys Asn Ala Asp Ala Asn Pro Pro ProPhe Lys Ser Val Trp 290 295 300 agc agg ttg gat gga caa tgg cct gat ggttta ttg gcg tca gat aat 960 Ser Arg Leu Asp Gly Gln Trp Pro Asp Gly LeuLeu Ala Ser Asp Asn 305 310 315 320 act ctt cat ttt gtc cat cca ttg actgtc aat tat tct ggc gtt tat 1008 Thr Leu His Phe Val His Pro Leu Thr ValAsn Tyr Ser Gly Val Tyr 325 330 335 gtc tgt aaa gta tca aat tcc ctt ggtcaa aga agt gat caa aag gtt 1056 Val Cys Lys Val Ser Asn Ser Leu Gly GlnArg Ser Asp Gln Lys Val 340 345 350 atc tac att tca gac atc ccg ctt acgcag acc tca tcc ata gca gtg 1104 Ile Tyr Ile Ser Asp Ile Pro Leu Thr GlnThr Ser Ser Ile Ala Val 355 360 365 gct gga gcc gtg att gga gct gtc ctggcc ctc ttc atc atc acc gtc 1152 Ala Gly Ala Val Ile Gly Ala Val Leu AlaLeu Phe Ile Ile Thr Val 370 375 380 ttt gtg act gtg ttg ctg acg cct cggaaa aag aga ccg tcc tat ctt 1200 Phe Val Thr Val Leu Leu Thr Pro Arg LysLys Arg Pro Ser Tyr Leu 385 390 395 400 gac aaa gta atc gac ctt cca cctaca cat aag cca ccc cct gta tat 1248 Asp Lys Val Ile Asp Leu Pro Pro ThrHis Lys Pro Pro Pro Val Tyr 405 410 415 gaa gaa cga att cct tct ctc cctcag aaa gac ctt ctt ggc cag act 1296 Glu Glu Arg Ile Pro Ser Leu Pro GlnLys Asp Leu Leu Gly Gln Thr 420 425 430 gaa cac ttg cct ttg cag act cagttc aag gag aaa gga gct ggt ggt 1344 Glu His Leu Pro Leu Gln Thr Gln PheLys Glu Lys Gly Ala Gly Gly 435 440 445 ctt cag ccc tct aat gga cca attagc agg aga ttt gac tat gag gat 1392 Leu Gln Pro Ser Asn Gly Pro Ile SerArg Arg Phe Asp Tyr Glu Asp 450 455 460 gag agc aca atg caa gaa gat ggaact cag cgc atg tgc ccc ctt tat 1440 Glu Ser Thr Met Gln Glu Asp Gly ThrGln Arg Met Cys Pro Leu Tyr 465 470 475 480 agc cag atg tgc cac caa gaccga agc cct cgc caa cat cac cca cgc 1488 Ser Gln Met Cys His Gln Asp ArgSer Pro Arg Gln His His Pro Arg 485 490 495 aac ccc gag aga ctc tac atcaac cca cga gaa cat tat gtg tga 1533 Asn Pro Glu Arg Leu Tyr Ile Asn ProArg Glu His Tyr Val 500 505 510 <210> SEQ ID NO 4 <211> LENGTH: 510<212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 4 Met Ala Arg ThrPro Gly Pro Ala Pro Leu Cys Pro Gly Gly Gly Lys 1 5 10 15 Ala Gln LeuSer Ser Ala Phe Pro Pro Ala Ala Gly Leu Leu Leu Pro 20 25 30 Ala Pro ThrPro Pro Pro Leu Leu Leu Leu Leu Ile Pro Leu Leu Leu 35 40 45 Phe Ser ArgLeu Cys Gly Ala Leu Ala Gly Ser Ile Ile Val Glu Pro 50 55 60 His Val ThrAla Val Trp Gly Lys Asn Val Ser Leu Lys Cys Leu Ile 65 70 75 80 Glu ValAsn Glu Thr Ile Thr Gln Ile Ser Trp Glu Lys Ile His Gly 85 90 95 Lys SerThr Gln Thr Val Ala Val His His Pro Gln Tyr Gly Phe Ser 100 105 110 ValGln Gly Asp Tyr Gln Gly Arg Val Leu Phe Lys Asn Tyr Ser Leu 115 120 125Asn Asp Ala Thr Ile Thr Leu His Asn Ile Gly Phe Ser Asp Ser Gly 130 135140 Lys Tyr Ile Cys Lys Ala Val Thr Phe Pro Leu Gly Asn Ala Gln Ser 145150 155 160 Ser Thr Thr Val Thr Val Leu Val Glu Pro Thr Val Ser Leu IleLys 165 170 175 Gly Pro Asp Ser Leu Ile Asp Gly Gly Asn Glu Thr Val AlaAla Val 180 185 190 Cys Val Ala Ala Thr Gly Lys Pro Val Ala Gln Ile AspTrp Glu Gly 195 200 205 Asp Leu Gly Glu Met Glu Ser Ser Thr Thr Ser PhePro Asn Glu Thr 210 215 220 Ala Thr Ile Val Ser Gln Tyr Lys Leu Phe ProThr Arg Phe Ala Arg 225 230 235 240 Gly Arg Arg Ile Thr Cys Val Val LysHis Pro Ala Leu Glu Lys Asp 245 250 255 Ile Arg Tyr Ser Phe Ile Leu AspIle Gln Tyr Ala Pro Glu Val Ser 260 265 270 Val Thr Gly Tyr Asp Gly AsnTrp Phe Val Gly Arg Lys Gly Val Asn 275 280 285 Leu Lys Cys Asn Ala AspAla Asn Pro Pro Pro Phe Lys Ser Val Trp 290 295 300 Ser Arg Leu Asp GlyGln Trp Pro Asp Gly Leu Leu Ala Ser Asp Asn 305 310 315 320 Thr Leu HisPhe Val His Pro Leu Thr Val Asn Tyr Ser Gly Val Tyr 325 330 335 Val CysLys Val Ser Asn Ser Leu Gly Gln Arg Ser Asp Gln Lys Val 340 345 350 IleTyr Ile Ser Asp Ile Pro Leu Thr Gln Thr Ser Ser Ile Ala Val 355 360 365Ala Gly Ala Val Ile Gly Ala Val Leu Ala Leu Phe Ile Ile Thr Val 370 375380 Phe Val Thr Val Leu Leu Thr Pro Arg Lys Lys Arg Pro Ser Tyr Leu 385390 395 400 Asp Lys Val Ile Asp Leu Pro Pro Thr His Lys Pro Pro Pro ValTyr 405 410 415 Glu Glu Arg Ile Pro Ser Leu Pro Gln Lys Asp Leu Leu GlyGln Thr 420 425 430 Glu His Leu Pro Leu Gln Thr Gln Phe Lys Glu Lys GlyAla Gly Gly 435 440 445 Leu Gln Pro Ser Asn Gly Pro Ile Ser Arg Arg PheAsp Tyr Glu Asp 450 455 460 Glu Ser Thr Met Gln Glu Asp Gly Thr Gln ArgMet Cys Pro Leu Tyr 465 470 475 480 Ser Gln Met Cys His Gln Asp Arg SerPro Arg Gln His His Pro Arg 485 490 495 Asn Pro Glu Arg Leu Tyr Ile AsnPro Arg Glu His Tyr Val 500 505 510 <210> SEQ ID NO 5 <211> LENGTH: 1317<212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(1317) <300> PUBLICATION INFORMATION: <301>AUTHORS: Satoh-Horikawa K. et al. <302> TITLE: Nectin-3, a new member ofimmunoglobulin-like cell adhesion molecules that shows homophilic andheterophilic cell-cell adhesion activities. <303> JOURNAL: J. Biol.Chem. <304> VOLUME: 275 <305> ISSUE: 14 <306> PAGES: 10291-10299 <307>DATE: 2000-04-07 <308> DATABASE ACCESSION NUMBER: GenBank accession No.AF195835 <309> DATABASE ENTRY DATE: 2000-04-13 <400> SEQUENCE: 5 atg gcgcgg acc ccg ggc ccg gcc ccg ttg tgt cct gga ggc ggc aaa 48 Met Ala ArgThr Pro Gly Pro Ala Pro Leu Cys Pro Gly Gly Gly Lys 1 5 10 15 gca caactt tcc tcg gcg ttt cct ccc gcg gcc gga ctg ctg ctg ccg 96 Ala Gln LeuSer Ser Ala Phe Pro Pro Ala Ala Gly Leu Leu Leu Pro 20 25 30 gcc ccg acgccg ccg ccg ctg ctg ctg ctg ctt att ccc ctg ctt ctc 144 Ala Pro Thr ProPro Pro Leu Leu Leu Leu Leu Ile Pro Leu Leu Leu 35 40 45 ttc tcc cgg ctctgt ggt gcc tta gct gga tca att att gtg gag cca 192 Phe Ser Arg Leu CysGly Ala Leu Ala Gly Ser Ile Ile Val Glu Pro 50 55 60 cat gtc aca gca gtgtgg gga aag aat gtt tca ttg aag tgt tta att 240 His Val Thr Ala Val TrpGly Lys Asn Val Ser Leu Lys Cys Leu Ile 65 70 75 80 gaa gtg aat gaa actata acc cag atc tca tgg gag aag ata cat ggc 288 Glu Val Asn Glu Thr IleThr Gln Ile Ser Trp Glu Lys Ile His Gly 85 90 95 aaa agt aca cag act gttgca gtt cat cat cct cag tat gga ttc tct 336 Lys Ser Thr Gln Thr Val AlaVal His His Pro Gln Tyr Gly Phe Ser 100 105 110 gtt caa gga gat tat caggga aga gtc ttg ttt aaa aac tat tca ctt 384 Val Gln Gly Asp Tyr Gln GlyArg Val Leu Phe Lys Asn Tyr Ser Leu 115 120 125 aat gat gca aca att actctg cat aac ata ggc ttc tca gat tct gga 432 Asn Asp Ala Thr Ile Thr LeuHis Asn Ile Gly Phe Ser Asp Ser Gly 130 135 140 aaa tat ata tgc aaa gccgtt aca ttc cca ctt gga aat gct cag tcc 480 Lys Tyr Ile Cys Lys Ala ValThr Phe Pro Leu Gly Asn Ala Gln Ser 145 150 155 160 tct aca aca gtg actgtg tta gtt gaa ccc aca gtg agc ctg ata aaa 528 Ser Thr Thr Val Thr ValLeu Val Glu Pro Thr Val Ser Leu Ile Lys 165 170 175 ggg ccg gat tct ttaatt gat gga ggg aat gag aca gta gca gcc gtt 576 Gly Pro Asp Ser Leu IleAsp Gly Gly Asn Glu Thr Val Ala Ala Val 180 185 190 tgt gta gca gcc actgga aag cca gtc gca cag att gac tgg gaa ggt 624 Cys Val Ala Ala Thr GlyLys Pro Val Ala Gln Ile Asp Trp Glu Gly 195 200 205 gat ctt ggt gaa atggaa tct agt aca act tct ttt cct aat gaa aca 672 Asp Leu Gly Glu Met GluSer Ser Thr Thr Ser Phe Pro Asn Glu Thr 210 215 220 gca acg att gtc agccaa tac aag ctg ttt ccc aca aga ttt gct cga 720 Ala Thr Ile Val Ser GlnTyr Lys Leu Phe Pro Thr Arg Phe Ala Arg 225 230 235 240 gga agg cga attact tgt gtt gta aaa cat cca gcc tta gaa aag gac 768 Gly Arg Arg Ile ThrCys Val Val Lys His Pro Ala Leu Glu Lys Asp 245 250 255 att cgc tac tctttc ata cta gac ata cag tat gct cct gaa gtt tca 816 Ile Arg Tyr Ser PheIle Leu Asp Ile Gln Tyr Ala Pro Glu Val Ser 260 265 270 gta aca gga tatgat gga aat tgg ttc gtg gga aga aaa ggt gtt aac 864 Val Thr Gly Tyr AspGly Asn Trp Phe Val Gly Arg Lys Gly Val Asn 275 280 285 ctc aag tgt aatgct gat gca aac cct cca ccc ttc aag tcc gtg tgg 912 Leu Lys Cys Asn AlaAsp Ala Asn Pro Pro Pro Phe Lys Ser Val Trp 290 295 300 agc agg ttg gatgga caa tgg cct gat ggt tta ttg gcg tca gat aat 960 Ser Arg Leu Asp GlyGln Trp Pro Asp Gly Leu Leu Ala Ser Asp Asn 305 310 315 320 act ctt catttt gtc cat cca ttg act gtc aat tat tct ggc gtt tat 1008 Thr Leu His PheVal His Pro Leu Thr Val Asn Tyr Ser Gly Val Tyr 325 330 335 gtc tgt aaagta tca aat tcc ctt ggt caa aga agt gat caa aag gtt 1056 Val Cys Lys ValSer Asn Ser Leu Gly Gln Arg Ser Asp Gln Lys Val 340 345 350 atc tac atttca gac atc ccg ctt acg cag acc tca tcc ata gca gtg 1104 Ile Tyr Ile SerAsp Ile Pro Leu Thr Gln Thr Ser Ser Ile Ala Val 355 360 365 gct gga gccgtg att gga gct gtc ctg gcc ctc ttc atc atc acc gtc 1152 Ala Gly Ala ValIle Gly Ala Val Leu Ala Leu Phe Ile Ile Thr Val 370 375 380 ttt gtg actgtg ttg ctg acg cct cgg aaa aag aga ccg tcc tat ctt 1200 Phe Val Thr ValLeu Leu Thr Pro Arg Lys Lys Arg Pro Ser Tyr Leu 385 390 395 400 gac aaagta atc gac ctt cca cct aca cat aag cca ccc cct gta tat 1248 Asp Lys ValIle Asp Leu Pro Pro Thr His Lys Pro Pro Pro Val Tyr 405 410 415 gaa gaacga att cct tct ctc cct cag aaa gac ctt ctt ggc cag gta 1296 Glu Glu ArgIle Pro Ser Leu Pro Gln Lys Asp Leu Leu Gly Gln Val 420 425 430 cgt gctctc gaa gac act taa 1317 Arg Ala Leu Glu Asp Thr 435 <210> SEQ ID NO 6<211> LENGTH: 438 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE:6 Met Ala Arg Thr Pro Gly Pro Ala Pro Leu Cys Pro Gly Gly Gly Lys 1 5 1015 Ala Gln Leu Ser Ser Ala Phe Pro Pro Ala Ala Gly Leu Leu Leu Pro 20 2530 Ala Pro Thr Pro Pro Pro Leu Leu Leu Leu Leu Ile Pro Leu Leu Leu 35 4045 Phe Ser Arg Leu Cys Gly Ala Leu Ala Gly Ser Ile Ile Val Glu Pro 50 5560 His Val Thr Ala Val Trp Gly Lys Asn Val Ser Leu Lys Cys Leu Ile 65 7075 80 Glu Val Asn Glu Thr Ile Thr Gln Ile Ser Trp Glu Lys Ile His Gly 8590 95 Lys Ser Thr Gln Thr Val Ala Val His His Pro Gln Tyr Gly Phe Ser100 105 110 Val Gln Gly Asp Tyr Gln Gly Arg Val Leu Phe Lys Asn Tyr SerLeu 115 120 125 Asn Asp Ala Thr Ile Thr Leu His Asn Ile Gly Phe Ser AspSer Gly 130 135 140 Lys Tyr Ile Cys Lys Ala Val Thr Phe Pro Leu Gly AsnAla Gln Ser 145 150 155 160 Ser Thr Thr Val Thr Val Leu Val Glu Pro ThrVal Ser Leu Ile Lys 165 170 175 Gly Pro Asp Ser Leu Ile Asp Gly Gly AsnGlu Thr Val Ala Ala Val 180 185 190 Cys Val Ala Ala Thr Gly Lys Pro ValAla Gln Ile Asp Trp Glu Gly 195 200 205 Asp Leu Gly Glu Met Glu Ser SerThr Thr Ser Phe Pro Asn Glu Thr 210 215 220 Ala Thr Ile Val Ser Gln TyrLys Leu Phe Pro Thr Arg Phe Ala Arg 225 230 235 240 Gly Arg Arg Ile ThrCys Val Val Lys His Pro Ala Leu Glu Lys Asp 245 250 255 Ile Arg Tyr SerPhe Ile Leu Asp Ile Gln Tyr Ala Pro Glu Val Ser 260 265 270 Val Thr GlyTyr Asp Gly Asn Trp Phe Val Gly Arg Lys Gly Val Asn 275 280 285 Leu LysCys Asn Ala Asp Ala Asn Pro Pro Pro Phe Lys Ser Val Trp 290 295 300 SerArg Leu Asp Gly Gln Trp Pro Asp Gly Leu Leu Ala Ser Asp Asn 305 310 315320 Thr Leu His Phe Val His Pro Leu Thr Val Asn Tyr Ser Gly Val Tyr 325330 335 Val Cys Lys Val Ser Asn Ser Leu Gly Gln Arg Ser Asp Gln Lys Val340 345 350 Ile Tyr Ile Ser Asp Ile Pro Leu Thr Gln Thr Ser Ser Ile AlaVal 355 360 365 Ala Gly Ala Val Ile Gly Ala Val Leu Ala Leu Phe Ile IleThr Val 370 375 380 Phe Val Thr Val Leu Leu Thr Pro Arg Lys Lys Arg ProSer Tyr Leu 385 390 395 400 Asp Lys Val Ile Asp Leu Pro Pro Thr His LysPro Pro Pro Val Tyr 405 410 415 Glu Glu Arg Ile Pro Ser Leu Pro Gln LysAsp Leu Leu Gly Gln Val 420 425 430 Arg Ala Leu Glu Asp Thr 435 <210>SEQ ID NO 7 <211> LENGTH: 2178 <212> TYPE: DNA <213> ORGANISM: Mouse<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (197)..(1846) <300>PUBLICATION INFORMATION: <301> AUTHORS: Satoh-Horikawa K. et al. <302>TITLE: Nectin-3, a new member of immunoglobulin-like cell adhesionmolecules that shows homophilic and heterophilic cell-cell adhesionactivities. <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 275 <305> ISSUE:14 <306> PAGES: 10291-10299 <307> DATE: 2000-04-07 <308> DATABASEACCESSION NUMBER: GenBank accession No. AF195833 <309> DATABASE ENTRYDATE: 2000-04-13 <400> SEQUENCE: 7 gaattcggca cgagcgacgg cggagtcgaggcagccgcga gcgctcggcc gagtggcggc 60 gggcggcgac ggcgcaggag ccgggggttgaggacacgcg cgctggccct tccgcgccgc 120 ggccgccgcc gccgccgcca cccagagcctgaggcgccgg ggcgcgggcg agcgggtggg 180 ccggggcaag gcgggc atg gcg cgg accccg ggc ccg gcc ccg ttg tgt cct 232 Met Ala Arg Thr Pro Gly Pro Ala ProLeu Cys Pro 1 5 10 gga ggc ggc aaa gca caa ctt tcc tcg gcg ttt cct cccgcg gcc gga 280 Gly Gly Gly Lys Ala Gln Leu Ser Ser Ala Phe Pro Pro AlaAla Gly 15 20 25 ctg ctg ctg ccg gcc ccg acg ccg ccg ccg ctg ctg ctg ctgctt att 328 Leu Leu Leu Pro Ala Pro Thr Pro Pro Pro Leu Leu Leu Leu LeuIle 30 35 40 ccc ctg ctt ctc ttc tcc cgg ctc tgt ggt gcc tta gct gga tcaatt 376 Pro Leu Leu Leu Phe Ser Arg Leu Cys Gly Ala Leu Ala Gly Ser Ile45 50 55 60 att gtg gag cca cat gtc aca gca gtg tgg gga aag aat gtt tcattg 424 Ile Val Glu Pro His Val Thr Ala Val Trp Gly Lys Asn Val Ser Leu65 70 75 aag tgt tta att gaa gtg aat gaa act ata acc cag atc tca tgg gag472 Lys Cys Leu Ile Glu Val Asn Glu Thr Ile Thr Gln Ile Ser Trp Glu 8085 90 aag ata cat ggc aaa agt aca cag act gtt gca gtt cat cat cct cag520 Lys Ile His Gly Lys Ser Thr Gln Thr Val Ala Val His His Pro Gln 95100 105 tat gga ttc tct gtt caa gga gat tat cag gga aga gtc ttg ttt aaa568 Tyr Gly Phe Ser Val Gln Gly Asp Tyr Gln Gly Arg Val Leu Phe Lys 110115 120 aac tat tca ctt aat gat gca aca att act ctg cat aac ata ggc ttc616 Asn Tyr Ser Leu Asn Asp Ala Thr Ile Thr Leu His Asn Ile Gly Phe 125130 135 140 tca gat tct gga aaa tat ata tgc aaa gcc gtt aca ttc cca cttgga 664 Ser Asp Ser Gly Lys Tyr Ile Cys Lys Ala Val Thr Phe Pro Leu Gly145 150 155 aat gct cag tcc tct aca aca gtg act gtg tta gtt gaa ccc acagtg 712 Asn Ala Gln Ser Ser Thr Thr Val Thr Val Leu Val Glu Pro Thr Val160 165 170 agc ctg ata aaa ggg ccg gat tct tta att gat gga ggg aat gagaca 760 Ser Leu Ile Lys Gly Pro Asp Ser Leu Ile Asp Gly Gly Asn Glu Thr175 180 185 gta gca gcc gtt tgt gta gca gcc act gga aag cca gtc gca cagatt 808 Val Ala Ala Val Cys Val Ala Ala Thr Gly Lys Pro Val Ala Gln Ile190 195 200 gac tgg gaa ggt gat ctt ggt gaa atg gaa tct agt aca act tctttt 856 Asp Trp Glu Gly Asp Leu Gly Glu Met Glu Ser Ser Thr Thr Ser Phe205 210 215 220 cct aat gaa aca gca acg att gtc agc caa tac aag ctg tttccc aca 904 Pro Asn Glu Thr Ala Thr Ile Val Ser Gln Tyr Lys Leu Phe ProThr 225 230 235 aga ttt gct cga gga agg cga att act tgt gtt gta aaa catcca gcc 952 Arg Phe Ala Arg Gly Arg Arg Ile Thr Cys Val Val Lys His ProAla 240 245 250 tta gaa aag gac att cgc tac tct ttc ata cta gac ata cagtat gct 1000 Leu Glu Lys Asp Ile Arg Tyr Ser Phe Ile Leu Asp Ile Gln TyrAla 255 260 265 cct gaa gtt tca gta aca gga tat gat gga aat tgg ttc gtggga aga 1048 Pro Glu Val Ser Val Thr Gly Tyr Asp Gly Asn Trp Phe Val GlyArg 270 275 280 aaa ggt gtt aac ctc aag tgt aat gct gat gca aac cct ccaccc ttc 1096 Lys Gly Val Asn Leu Lys Cys Asn Ala Asp Ala Asn Pro Pro ProPhe 285 290 295 300 aag tcc gtg tgg agc agg ttg gat gga caa tgg cct gatggt tta ttg 1144 Lys Ser Val Trp Ser Arg Leu Asp Gly Gln Trp Pro Asp GlyLeu Leu 305 310 315 gcg tca gat aat act ctt cat ttt gtc cat cca ttg actgtc aat tat 1192 Ala Ser Asp Asn Thr Leu His Phe Val His Pro Leu Thr ValAsn Tyr 320 325 330 tct ggc gtt tat gtc tgt aaa gta tca aat tcc ctt ggtcaa aga agt 1240 Ser Gly Val Tyr Val Cys Lys Val Ser Asn Ser Leu Gly GlnArg Ser 335 340 345 gat caa aag gtt atc tac att tca gat cct cct acc accacc acc ctt 1288 Asp Gln Lys Val Ile Tyr Ile Ser Asp Pro Pro Thr Thr ThrThr Leu 350 355 360 cag ccg aca gtt cag tgg cat tcc tca cct gct gac gtccag gat ata 1336 Gln Pro Thr Val Gln Trp His Ser Ser Pro Ala Asp Val GlnAsp Ile 365 370 375 380 gca aca gag cat aaa aaa ttg ccc ttt cct ttg tcaact ttg gca aca 1384 Ala Thr Glu His Lys Lys Leu Pro Phe Pro Leu Ser ThrLeu Ala Thr 385 390 395 ctt aag gat gac aca att ggc acc atc att gct agtgta gtg ggt ggg 1432 Leu Lys Asp Asp Thr Ile Gly Thr Ile Ile Ala Ser ValVal Gly Gly 400 405 410 gct ctc ttc tta gtg ctt gtg agc att tta gct ggggta ttc tgc tat 1480 Ala Leu Phe Leu Val Leu Val Ser Ile Leu Ala Gly ValPhe Cys Tyr 415 420 425 agg aga cga cgg acg ttt cgt gga gac tac ttt gccaaa aac tac att 1528 Arg Arg Arg Arg Thr Phe Arg Gly Asp Tyr Phe Ala LysAsn Tyr Ile 430 435 440 cca cca tca gac atg cag aaa gaa tca cag att gatgtt ctt cac cag 1576 Pro Pro Ser Asp Met Gln Lys Glu Ser Gln Ile Asp ValLeu His Gln 445 450 455 460 gat gag ctg gat tct tac cca gac agt gta aaaaag gaa aac aaa aat 1624 Asp Glu Leu Asp Ser Tyr Pro Asp Ser Val Lys LysGlu Asn Lys Asn 465 470 475 cca gta aac aac ctg atc cgc aaa gac tac ttagag gag cct gag aaa 1672 Pro Val Asn Asn Leu Ile Arg Lys Asp Tyr Leu GluGlu Pro Glu Lys 480 485 490 act cag tgg aat aat gta gag aac ctc act aggttt gaa aga ccg atg 1720 Thr Gln Trp Asn Asn Val Glu Asn Leu Thr Arg PheGlu Arg Pro Met 495 500 505 gat tac tat gaa gat cta aaa atg gga atg aagttt gtc agt gat gaa 1768 Asp Tyr Tyr Glu Asp Leu Lys Met Gly Met Lys PheVal Ser Asp Glu 510 515 520 cgc tac aat gaa agt gaa gat ggt ttg gtt tctcat gta gat ggc tcc 1816 Arg Tyr Asn Glu Ser Glu Asp Gly Leu Val Ser HisVal Asp Gly Ser 525 530 535 540 gta att tcc agg agg gag tgg tat gtc taacagccactga cgcgacttca 1866 Val Ile Ser Arg Arg Glu Trp Tyr Val 545ctatgtacaa ggtttcattc acactagttg accattttca gattgttcat actttttctt 1926gaggaagaat aagctttttc aagttgattt cgagcttact ttttatattc tgatctgaca 1986aatgaaaatg taaaacctgg gttcaatgta tctgagctgc tttacagttt tcactgctat 2046actactgtct caagatttaa attctaatgc agagtacttt attggtctga ggcacacagg 2106taagaaagat gtcaacgtta aatgtatgac gtttttggta caaaaattaa aaaaaaaaaa 2166aaaaaactcg ag 2178 <210> SEQ ID NO 8 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: C-Terminal Sequence ofNectin-1alpha <400> SEQUENCE: 8 Ser Phe Ile Ser Lys Lys Glu Trp Tyr Val1 5 10 <210> SEQ ID NO 9 <211> LENGTH: 10 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: C-Terminal Sequence of Nectin-1beta<400> SEQUENCE: 9 Val Arg Thr Thr Glu Pro Arg Gly Glu Cys 1 5 10 <210>SEQ ID NO 10 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: C-Terminal Sequence of Nectin-2alpha <400>SEQUENCE: 10 Ser Leu Ile Ser Arg Arg Ala Val Tyr Val 1 5 10 <210> SEQ IDNO 11 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: C-Terminal Sequence of Nectin-2delta <400>SEQUENCE: 11 Asp Glu Phe Val Ser Arg Ala Met Tyr Val 1 5 10 <210> SEQ IDNO 12 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: C-Terminal Sequence of Nectin-3alpha <400>SEQUENCE: 12 Ser Val Ile Ser Arg Arg Glu Trp Tyr Val 1 5 10 <210> SEQ IDNO 13 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: C-Terminal Sequence of Nectin-3beta <400> SEQUENCE:13 Leu Tyr Ile Asn Pro Arg Glu His Tyr Val 1 5 10 <210> SEQ ID NO 14<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: C-Terminal Sequence of Nectin-2gamma <400> SEQUENCE: 14 LeuGly Gln Val Arg Ala Leu Glu Asp Thr 1 5 10

1. A protein nectin-3 having the amino acid sequence of SEQ ID NO:
 2. 2.A protein nectin-3 having the amino acid sequence of SEQ ID NO:
 4. 3. Aprotein nectin-3 having the amino acid sequence of SEQ ID NO:
 6. 4. Apolynucleotide encoding the protein nectin-3 of any of claims 1-3. 5.The polynucleotide as claimed in claim 2, which has the base sequence ofSEQ ID NO:
 1. 6. The polynucleotide as claimed in claim 2, which has thebase sequence of SEQ ID NO:
 3. 7. The polynucleotide as claimed in claim2, which has the base sequence of SEQ ID NO:
 5. 8. A recombinant vectorhaving the polynucleotide of any of claims 4 to
 7. 9. An antibodyagainst the protein nectin-3 of any of claims 1 to 3.